Fibrous fibrin sheet and method for producing same

ABSTRACT

Fibrin in sheet form is prepared by centrifuging an aqueous dispersion of monomeric fibrin under fibrin-polymerizing conditions. The centrifugation is conducted in a vessel having a wall for intercepting centrifuged particles and at a speed pelletizing the resulting strands of polymerized fibrin thereon. The pelletized strands interlock to form a fibrous sheet, which is removed from the vessel.

United States Patent 91 Breillatt, Jr.

1541 FIBROUS FIBRIN SHEET AND METHOD FOR PRODUCING SAME [75] Inventor:Julian P. Breillatt, Jr., Oak Ridge,

Tenn.

[73] Assignee: The United States of America as represented by the UnitedStates Atomic Energy Commission 221 Filed: Jan. 18,1971

21 Appl.No.: 107,382

[52] US. Cl. ..162/151, 128/156, 128/325, l28/335.5, 264/311, 424/14,424/15, 424/28,

[51] Int. Cl. ..D2lh 5/20 [58] Field of Search ..162/151, 143, 384;106/124, 106/161; 128/156,325,335.5;424/14, 15, 27, 28; 264/311; 260/112R, 112 B [56] ReierencesCited UNITED STATES PATENTS 3,617,443 11/1971Chleq ..162/384 X 3,210,241 10/1965 Grauss et al. 3,036,341 5/1962Taylor ..264/311 X [451 Mar. 27, 1973 3,014,024 12/1961 Lieberman et a]..264/311 X 2,533,004 12/1950 Ferry et al. ..128/156 X 2,992,882 7/1961Bcsso ct al ..106/124 X 2,385,802 10/1945 Ferry ..106/124 3,523,8078/1970 Gerendas 1215/3355 X OTHER PUBLICATIONS Harvey, The Use of FibrinPaper and Forms in Surgery, Boston Medical and Surgical Journal; Vol.174, No. 18; pp. 658-59, May 4,1916.

Primary Examiner-S. Leon Bashore Assistant ExaminerFrederick FreiAttorney-Roland A. Anderson [57] ABSTRACT 8 Claims, 1 Drawing FigurePATENTEDMARZY m5 INVENTOR. Julian I? BmTIIaH, Jr.

ATTORQIEY.

FIBROUS FIBRIN SHEET AND METHOD FOR PRODUCING SAME BACKGROUND OF THEINVENTION This invention was made in the course of, or under, a contractwith the united States Atomic Energy Commission.

Human fibrin is an immunologically neutral body protein which is findingincreasing application as a 1 research tool and a therapeutic material.For example, U.S. Pat. No. 3,523,807 describes a process wherein amixture of powdered human fibrin and water is molded to form a shapedprosthetic appliance; after treatment with a cross-linking agent, theappliance is used in surgical procedures. U.S. Pat. No. 2,385,802describes the production of plastics by mixing powdered fibrinogen witha plasticizer, moldingv the resulting mixture, and setting the moldedarticle by heat. U.S. Pat. No. 2,385,803 relates to preparing proteinplastics by mixing powdered fibrinogen with a selected proteinand aplasticizer, and setting the resulting mixture under heat and pressure.British Patent 866,628 relates to the preparation of collagen films bycentrifuging an aqueous acid dispersion of collagen fibers in a basketcentrifuge lined with a filter medium. The centrifuge is operated at aspeed distributing the dispersion over the face of the filter and thenat a speed expelling the dispersing liquid through the filter. Thecollagen fibers trapped on the'face of the filter form a removable film.

Sheet material containing fibrin is especially suitable for certainapplications. In surgery, for instance, sheets containing preformedfibrin foam mixed with thrombin are used to promote hemostasis. Fibrinfilms produced by pressing and flattening blood clots have been used forplatelet function tests. Fibrin in sheet form also may find use as adressing for burns, a membrane for the retention of body fluids, and asubstrate for the growth of cells.

The known methods of forming fibrin-containing sheets are tedious andare not well adapted for the production, on a reproducible basis, ofsheets having a closely controlled thickness and a comparatively largearea.

It is, therefore, an object of this invention to provide an improvedmethod for the production of fibrin in the form of sheets.

It is another object of this invention to provide a method forreproducibly producing fibrin sheets, said method providing closecontrol of the thickness of said sheets.

It is another object to provide a method for producing fibrin in theform of sheets having a comparatively large area.

It is another object to provide fibrin in the form of a sheet ofinterlocked fibrin fibers extending generally in the plane of the sheet.

Other objects of this invention will be made apparent hereinafter.

SUMMARY OF THE INVENTION v centrifugal acceleration therein and at aspeed pelletizing on said wall the strands of fibrin resulting from theBRIEF DESCRIPTION OF THE DRAWING The single FIGURE is a photomicrograph(total magnification: 30,000X) of an interior portion of a 0 fibrinsheet produced in accordance with this invention. The portion shown inthe FIGURE was obtained by sectioning the sheet along planes parallel tothe faces of the sheet.

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the preferred form of myinvention, an aqueous dispersion of monomeric fibrin molecules iscentrifuged under fibrin-polymerizing conditions in a highspeed,bowl-type rotor of comparatively large volume. One such rotor is theso-called B-XV rotor described in detail in the following reference:Anderson, N. G., et al, Analytical Techniques for Cell Fractionations V:Characteristics of the B-XIV and B-XV Zonal Centrifuge Rotors,Analytical Biochemistry 21, 235 (1967). Another suitable rotor is theTidesign (Beckman Instruments, Inc'.). These rotors contain radiallyoriented partitions, or septa, which are secured to the rotor core andextend close to the rotor wall. The rotors need not, however, beprovided with septa to accomplish the purposes of this invention.

The above-mentioned dispersion of monomeric fibrin may be provided invarious forms known to the art. It may, for example, be in the form of avolume of whole blood plasma, or a selected fraction thereof, in whichmonomeric fibrin is being formed as a result of induction of theblood-clotting reaction. As another illustration, it may be in the formof an aqueous solution 40 of purified fibrinogen and a divalent alkalineearth cation (e.g., Ca, Mg) containing a sufficient quantity of aprotolytic enzyme (e.g., thrombin, trypsin) to induce thefibrinogen-to-monomeric-fibrin reaction. Preferably, the pH of thesolution in which the monomeric fibrin is being formed is adjusted to avalue in the range of about 5.5 to 10.5, if it is not already in thatrange, in order to promote the ensuing polymerization of monomericfibrin molecules. The formation of monomeric and polymerized fibrin isdiscussed in various texts, such as The Plasma Proteins, Vol. II, NewYork: Academic Press, 1960.

The following is a general illustration of my method as applied to thepreparation of fibrin sheet from freshfrozen human plasma which has beentreated with a calcium-complexing agent, such as sodium citrate, toinhibit fibrin formation during storage. Citrated plasma is thawed andthen is centrifuged at a comparatively low speed to removecryoglobulins, which otherwise would deposit in the product sheet.Removal of the cryoglobulins is conducted at temperatures in the regionof 5C, since at higher temperatures the cryoglobulins become soluble.The resulting clarified, cool plasma is then treated to induce thenormal bloodclotting reaction. This may be accomplished by the additionof calcium ions, as by the addition of an aqueous solution of calciumchloride or calcium acetate. The calcium ions are added somewhat inexcess of the amount required to saturate the available citratecalcium-binding sites. As is well known in the art, this addition ofcalcium induces the formation of monomeric fibrin molecules; normally,these molecules combine promptly by polymerization to form insolublefibrin strands which in turn combine to form a gel, or clot. The rate ofpolymerization of the monomeric fibrin is temperature dependent and isrelatively low at temperatures from just above the freezing point of themixture to, say, 5C.

Following the reaction-inducing procedure just described, the plasma isloaded into the centrifuge rotor, and the latter is spun at pelletizingspeed while polymerization is taking place. The term pelletizing speed"as used here refers to a speed of centrifugation generating sufficientcentrifugal force to pelletize, or deposit, on the rotor wall the fibrinstrands formed by polymerization. To avoid gelling of the plasma, Iprefer to retard the polymerization reaction initially by maintainingthe plasma at a temperature in a first selected range; I maintain thistemperature from the time that clotting is induced as described untilthe centrifuge rotor is in the region of the pelletizing speed. I preferto conduct the pelletizing operation itself at a temperature in a secondselected range higher than said first range in order to accelerate thepolymerization reaction forming the fibrin strands. This can beaccomplished conveniently by permitting the plasma to warm to the secondselected temperature as the rotor approaches pelletizing speed. AlthoughI increase the temperature of the plasma to promote polymerizationduring pelletizing, gelling is negligible because of the separativeeffect of the centrifugal field. In other words, the blood-clottingreaction is taking place while the rotor is at pelletizing speed, butthe usual blood clot is not produced because of the action of thecentrifugal field.

Duringpelletizing I maintain the plasma below the temperature at whichfibrin denatures appreciably. In the typical pelletizing operation,considerable heat is imparted to the plasma from the rotor wall;consequently, during pelletizing I prefer to cool the plasma as requiredto maintain its temperature above about C and below about 55C. Attemperatures below about 15C the pelletizing time required to formsheets is impractically long. At temperatures above about 55C,denaturing of the protein occurs to an objectionable extent.

At pelletizing speed, the polymerized fibrin strands are impelled uponthe rotor wall, where they form a layer and interlock by mechanical andperhaps chemical interaction to form a continuous sheet. Some monomericfibrin particulates also will be pelletized on the wall, and most ofthese will polymerize in place in the course of the pelletizingoperation, becoming an integral part of the resulting sheet. Thepelletizing operation is continued for a period sufficient to form asheet of the desired thickness, as predetermined empirically or bycalculation.

Following the centrifuge operation, the rotor is emptied. Theabove-mentioned sheet is recovered from the rotor wall, as by peeling itaway from the rotor. The recovered sheet, which normally is flexible,comprises a loose mesh, or fabric, of fibrin fibers which extendgenerally in the plane of the sheet. When prepared as just described,the sheet contains certain non-aggregated, non-polymerized proteins-suchas serum globulins and albuminwhich sediment into the sheet during thecentrifugation operation. These can be removed, if desired, by washingwith various selective solvents. The fibrin sheet produced as describedcan be used immediately or can be stored. Preferably, it is stored in0.85 weight percent sodium chloride solution, distilled water, or someother compatible liquid maintained at a temperature retarding bacterialgrowth.

The following is a more specific illustration of my process and theproduct obtained thereby.

EXAMPLEl Fresh-frozen citrated human blood plasma was thawed at 4C.Cryoglobulins were removed from the plasma by a comparatively low-speedcentrifugation (conducted at 1,500 rpm in a swinging-bucket rotor havinga diameter of about 10 inches). The clotting reaction then was inducedby stirring 34 ml of M CaCl into 1,125 ml of the centrifuged plasma, thelatter being at about 4C. (At 4C the clotting reaction goes tocompletion in roughly 48 hours.) The resulting mixture was promptlyloaded into a B-XV rotor having a removable seal permitting loadingduring rotation. The loading operation was conducted at 3,000 rpm and atabout 8C. After loading, the plasma was overlaid with an isotonic salinesolution to displace air from the rotor. The rotor speed then wasincreased to maximum value (=28,000 rpm) and maintained at approximatelythis value for a period of 28 hours; thus, during this period the plasmawas subjected to a total pelletizing force (a) t) of about 8.6 X 10sec". During the initial part of the centrifugation the plasma waspermitted to warm to 15C, and it was maintained at this temperature bycooling for the remainder of the run. At the end of the run, the liquidand the septa were removed from the rotor.

Examination of the rotor revealed that the material pelletized on thewall during centrifugation had formed a thin, integral sheet which wasco-extensive with the rotor wall. This sheet, or film, was peeled fromthe wall in the form of a transparent, flexible cylinder. After storagefor 12 hours in 0.85 percent sodium chloride solution, the sheetmeasured 56 cm. in circumference, 7.5 cm. in height, and 0.016 cm. inthickness. It was noted that the sheet swelled somewhat and becameincreasingly opaque while exposed to the storage medium. As shown in theaccompanying FIGURE, the unstressed sheet comprised a loose mesh offibers which occupied less than half the volume of the sheet. Thesurface of the fabric was smooth and amorphous, as observed by bothscanning electron microscopy and light microscopy. The typical fibrinfiber was oriented in the plane of the sheetthat is, comparatively fewfibers were oriented transversely with respect to the plane of thesheet. It was noted that the typical fiber was not flat, but wavy; thisaccounts for some of the apparent discontinuities in the fibers shown inthe accompanying FIGURE, since the sheet was sectioned along a planeparallel to the faces of the sheet. The sheet was of highly uniformthickness, as would be expected from centrifugal deposition on a uniformwall.

The tensile properties of the fibrin sheet, or fabric, were determinedwith a commercial machine (Model TTCL, lnstron Corporation, Canton,Mass). The ultimate stress was found to be 880 lbs/in? The stressstraincurve was similar to that of an elastomer.

It is not essential that cryoglobulins be removed from the plasma priorto the pelletizing operation. They may, if desired, be left in theplasma, in which case they will be deposited on the rotor wall duringcentrifugation and will in large part be incorporated in the resultingsheet. If desired, they may be removed from the fibrin sheet by washingthe sheet in a suitable solution, as in 25C aqueous 0.85 percent sodiumchloride.

EXAMPLE n i A B-XV rotor was loaded bysuccessive introduction of thefollowing through the edge line of the rotor: (1) 100 ml of salineoverlay, (2) a-mixture of 1,000 ml of clarified plasma and 30 'ml of 1 MCaCl (3) 540 ml of percent sucrose aqueous solution. As the rotor wasbrought to pelletizing speed, the sucrose solution formed an annularband, or layer, isolating the plasma from the rotor wall. Pelletizing ofthe polymerized fibrin strands was effected generally as described, withthe exception that the band of sucrose solution retarded thesedimentation of non-aggregated, nonpolymerized (i.e., lower-mass)proteins, such as albumin and serum globulins, so that comparatively fewof them reached the rotor wall.

The sheet removed from the rotor wall was generally similar to thatobtained in Example I, being a transparent, flexible, uniform cylinderhaving essentially the same dimensions. The sheet comprised a loose meshof interlocked fibrin fibers, the fibers being oriented generally in theplane of the sheet and occupying less than half the volume of thesheet.As compared with sheets produced in similar runs conducted without asucrose band, there was a marked reduction in the content of thecomparatively low-mass proteins, such as non-aggregated albumin andserum globulins.

. As mentioned, the dispersion of monomeric fibrin may be in variousforms, including an aqueous solution of purified fibrinogen wherein thefibrinogen-tomonomeric-fibrin reaction has been induced. The dispersionalso may be in the form of fresh blood plasma in which that reaction istaking place, although stringent precautions must be taken duringprocessing to avoid massive coagulation. My process is not limited I tothe production of fibrin sheet from human plasma but is applicable toother animal plasmas as well. It is wellknown thatthe clotting processis essentially the same in the various animal plasmas. The fibrin sheetsproduced from various solutions in which thefibrinogen-to-monomeric-fibrin reaction is taking place are generallysimilar and in most instances are suitable for use in the sameapplications. The sheets may not be identical, however. For example,unless certain agents such as cysteine are present, fibrin produced fromfibrinogen and thrombin is urea-soluble because of the absence of theusual fibrin-stabilizing factor.

Referring to the rotor used to centrifuge the dispersion of monomericfibrin, I prefer to employ a rotor of the bowl type. Such rotors lendthemselves to the production of sheets of various configurations; forexample, the bowl can be dimensioned to yield a fibrin sheet which aftercutting transversely is a square. A long bowl-type rotor of small radiuscan be used to produce the fibrin sheet in the form of tubing. My methodcan, however, be conducted in a sector-shaped vessel adapted to be swungabout an axis.

It is typical for the deposited fibrin sheet to conform to theconfiguration of the rotor wall, and thus the sheet formed in a standardbowl-type rotor is a smoothfaced cylinder, or sleeve, of essentiallyuniform thickness. If desired, however, the rotor wall may be configuredor patterned to form a product sheet having a special surface or shape.Furthermore, the rotor may be provided with a liner, such as looselywoven cellulose, into the interstices of which the centrifuged fibrinwill be pelletized, forming a composite structure characterized byenhanced strength or other special characteristics.

Referring to the processing of plasma in which clotting has beenprevented by an inhibitor, the clotting reaction may be induced by theaddition of an effective amount of various calcium compounds (other thanthe citrate) which are soluble in the plasma. As is well known, thecations of various divalent alkaline earths may be so used. Inductionalso may be effected by addition of thrombin or other appropriateprotolytic enzymes. If desired, induction may be deferred until theplasma is loaded into the centrifuge rotor while the latter is eitherstationary or rotating. Normal fresh plasma contains sufficient calciumions for the clotting reaction to begin almost immediately. The wordingplasma in which the fibrinogen-to-monomeric fibrin reaction has beeninduced is used herein to include fresh plasma or a fresh plasmafraction maintained under conditions (e.g., a suitable temperature)permitting that reaction to take place.

Referring to the pelletizing operation, it will be understood that thepelletizing speed as defined above encompasses a range of speeds. I havefound that pelletizing fields exceeding about 20,000 g are effective inproducing fibrin sheets in a practical time. It will be understood that,if desired, the rotor speed may be varied during the course of apelletizing operation. The length of time that the rotor is operated atpelletizing speed depends on such parameters as the diameter of therotor, the volume of plasma in the rotor, the temperature of the plasma,and the sheet thickness desired. Even the highest centrifuge speeds nowobtainable, such as those reached with analytical centrifuges, aresuitable.

As mentioned, the product sheet may be stored by suspending it invarious solutions. I have found that the fracture load for samples of mysheet stored in saline solution for seven days is about twice that forsimilar samples stored in distilled water for the same period. My fibrinsheets have been dried at room temperature and then rehydrated, withapparent retention of their initial elasticity and strength.

It was pointed out about that certain non-aggregated, non-polymerizedproteins (e.g., serum globulins) will sediment into the fibrin sheetduring pelletization and that these can be removed in a modified form ofmy method wherein the plasma is subjected to zonal centrifugation (seeThe Development of Zonal Centrifuges, National Cancer InstituteMonograph 21, June 1966). In another form of my method utilizing zonalcentrifugation, the fibrin strands centrifugally accelerated toward therotor wall are sedimented through one or more zones of liquid reagentsselected to modify the properties of the strands before they arepelletized on the wall (see US. Pat. No. 3,519,400, to N. G. Anderson).It will also be understood that the fibrin sheet formed on the rotorwall may be modified by various chemical treatments, either in situ orafter removal from the wall.

It will be apparent that my method for producing fibrin sheet is highlyreproducible, since both the dispersion being centrifuged and theconditions for centrifugation can be standardized to yield virtuallyidentical products from run to run.

Having thus described my invention, 1 claim:

1. The method of producing fibrin in sheet form which comprises thesteps of centrifuging an aqueous dispersion of fibrin wherein monomericfibrin molecules are combining by polymerization to form strands offibrin, said centrifuging step being conducted in a vessel having a wallfor interception of particles undergoing centrifugal accelerationtherein and at a speed pelletizing on said wall the strands of fibrinresulting from polymerization, wherein the strands so pelletizedinterlock to form a sheet, and recovering the resulting sheet from saidwall.

2. The method of claim 1 wherein said aqueous dispersion comprises wholeblood plasma.

3. The method of claim 1 wherein said aqueous dispersion comprises ablood plasma.

4. The method of claim 1 wherein said aqueous dispersion is provided inthe form of an aqueous solution of purified fibrinogen, a divalentalkaline earth cation, and a small but effective amount of a catalystfor the fibrinogen-to-monomeric-fibrin reaction.

5. The method of claim 1 wherein said aqueous dispersion is at a pH inthe range of 5.5 to 10.5.

6. The method of claim 1 wherein said centrifuging step is conducted ata temperature in the range of 15 to 55 C.

7. The method of claim 1 wherein said centrifuging step is conducted ata speed generating a pelletizing force of at least 20,000 g.

8. A sheet of fibrin produced by the process of claim 1.

2. The method of claim 1 wherein said aqueous dispersion comprises wholeblood plasma.
 3. The method of claim 1 wherein said aqueous dispersioncomprises a blood plasma.
 4. The method of claim 1 wherein said aqueousdispersion is provided in the form of an aqueous solution of purifiedfibrinogen, a divalent alkaline earth cation, and a small but effectiveamount of a catalyst for the fibrinogen-to-monomeric-fibrin reaction. 5.The method of claim 1 wherein said aqueous dispersion is at a pH in therange of 5.5 to 10.5.
 6. The method of claim 1 wherein said centrifugingstep is conducted at a temperature in the range of 15* to 55* C.
 7. Themethod of claim 1 wherein said centrifuging step is conducted at a speedgenerating a pelletizing force of at least 20,000 g.
 8. A sheet offibrin produced by the process of claim 1.